The initial response of osteoblasts to mechanical stimulation is a rapid rise in intracellular Ca2+ that is dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. We have demonstrated that Ca2+ entry via L-type voltage-sensitive Ca2+ channels (L-VSCC) is important to osteoblast proliferation, in vitro, and mechanically-induced bone formation, in vivo. We have also found that intracellular Ca2+ release through activation of phospholipase C/IP3 is responsible for shear-induced NFkB translocation and subsequent changes in gene expression. Finally, our data suggest that activation of phospholipase C by shear is mediated by activation of mechanically-sensitive channels and the L-VSCC that induces Ca2+ dependent nucleotide release. This release, in turn, results in activation of purinergic receptors (P2) to alter intracellular Ca2+ release. These data, coupled with other data from our lab, have led us to hypothesize that Ca2+ is elevated in discrete subcellular domains within the cell. In this scenario, extracellular Ca2+ entry would stimulate release of factors, such as ATP and/or UTP, that are important in signal amplification, while intracellular Ca2+ release via ATP activaton of P2 receptors would result in changes in gene expression. We present data suggesting the L-VSCC a1 isoform, Cav 1.2, is important in this response of osteoblasts to mechanical stimulation. To study the interaction of the L-VSCC with ATP release and the resulting activation of P2 receptors, we will use both cell biologic and in vivo genetic knockout studies to examine the role of the Cavl.2 a1 L-VSCC as well as P2 receptors in mechanotransduction. Our specific aims are to: 1) define the role of the L- VSCC Cavl.2 a1 subunit in the response of bone and osteoblasts to mechanical stimulation and 2) examine the effects of P2 receptor activation in the response of [Ca2+]i, prostaglandin secretion, transcription factor activation and changes in gene expression in osteoblastic cells subjected to fluid shear. Completion of these aims will provide added insight into the initial perception and rapid responses to mechanical stimulation of osteoblasts and may provide pharmacologic targets for alteration of bone formation.